Journal of Cell Science

cdc-25.2, a C. elegans ortholog of cdc25, is required to promote oocyte maturation.

Publication Date: 2010 Mar 15 PMID: 20200231
Authors: Kim, J. - Kawasaki, I. - Shim, Y. H.
Journal: J Cell Sci

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

post to: CiteULike

Chemotactic activation of Dictyostelium AGC-family kinases AKT and PKBR1 requires separate but coordinated functions of PDK1 and TORC2.

Publication Date: 2010 Mar 15 PMID: 20200230
Authors: Liao, X. H. - Buggey, J. - Kimmel, A. R.
Journal: J Cell Sci

Protein kinases AKT and PKBR1 of Dictyostelium belong to the AGC protein kinase superfamily. AKT and PKBR1 are phosphorylated at similar sites by phosphoinositide-dependent kinase 1 (PDK1) and TORC2 kinases; however, they have different subcellular localizing domains. AKT has a phosphoinositide 3-kinase (PI3K)/phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-regulated PH (pleckstrin homology) domain whereas PKBR1 is myristoylated and persistently membrane localized. Using strains defective for PI3K/PtdIns(3,4,5)P(3)-, PDK1- and TORC2-signaling or strains that express phospho-site mutants of AKT and PKBR1, we dissect the different roles of PI3K/PtdIns(3,4,5)P(3), PDK1 and TORC2. We show that activation of AKT and PKBR1 requires PDK1-site phosphorylation, but that phosphorylation by TORC2 is insufficient for AKT or PKBR1 activation. However, PDK1-site phosphorylation is dependent on phosphorylation by TORC2, which suggests that there is regulatory coordination among PDK1, TORC2 and their phospho-site targets. This defines a separate input for signaling in control of chemotaxis and dependency on PDK1 function. We also demonstrate that PDK1 in Dictyostelium functions independently of PI3K/PtdIns(3,4,5)P(3). Finally, we show that AKT and PKBR1 exhibit substrate selectivity and identify two novel lipid-interacting proteins preferentially phosphorylated by AKT. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a central role.

post to: CiteULike

Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation.

Publication Date: 2010 Mar 15 PMID: 20200229
Authors: Radtke, S. - Wuller, S. - Yang, X. P. - Lippok, B. E. - Mutze, B. - Mais, C. - de Leur, H. S. - Bode, J. G. - Gaestel, M. - Heinrich, P. C. - Behrmann, I. - Schaper, F. - Hermanns, H. M.
Journal: J Cell Sci

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

post to: CiteULike

Welcome to a new kind of tension: translating kinetochore mechanics into a wait-anaphase signal.

Publication Date: 2010 Mar 15 PMID: 20200228
Authors: Maresca, T. J. - Salmon, E. D.
Journal: J Cell Sci

Recent high-resolution studies of kinetochore structure have transformed the way researchers think about this crucial macro-molecular complex, which is essential for ensuring chromosome segregation occurs faithfully during cell division. Kinetochores mediate the interaction between chromosomes and the plus-ends of dynamic spindle microtubules and control the timing of anaphase onset by regulating the spindle assembly checkpoint (SAC). There is much debate in the SAC research community as to whether mitotic cells sense only microtubule attachment at the kinetochore, or both attachment and tension, before committing to anaphase. In this Commentary, we present a brief history of the tension-versus-attachment debate, summarize recent advances in our understanding of kinetochore structure and focus on the implications of a phenomenon known as intrakinetochore stretch for SAC regulation. We also hypothesize how intrakinetochore stretch might impact SAC function by regulating both microtubule attachment stability and the localization and activity of checkpoint components at the kinetochore.

post to: CiteULike

The architecture of an excitatory synapse.

Publication Date: 2010 Mar 15 PMID: 20200227
Authors: Chua, J. J. - Kindler, S. - Boyken, J. - Jahn, R.
Journal: J Cell Sci

post to: CiteULike

2009 winner: ravi desai.

Publication Date: 2010 Mar 15 PMID: 20200226
Authors: Watt, F.
Journal: J Cell Sci

post to: CiteULike

Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4.

Publication Date: 2010 Mar 2 PMID: 20197409
Authors: Kumeta, M. - Yoshimura, S. H. - Harata, M. - Takeyasu, K.
Journal: J Cell Sci

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

post to: CiteULike

MAPK scaffolding by BIT1 in the Golgi complex modulates stress resistance.

Publication Date: 2010 Mar 2 PMID: 20197408
Authors: Yi, P. - Nguyen, D. T. - Higa-Nishiyama, A. - Auguste, P. - Bouchecareilh, M. - Dominguez, M. - Bielmann, R. - Palcy, S. - Liu, J. F. - Chevet, E.
Journal: J Cell Sci

The endoplasmic reticulum (ER is an essential organelle whose major functions are to ensure proper secretory protein folding and trafficking. These mechanisms involve the activation of specific ER-resident molecular machines, which might be regulated by their membranous environments. Based on this observation, we aimed to characterize the proteome of ER-membrane microdomains to identify new components of the ER that have a role in secretory pathway-associated functions. Using this approach with dog pancreatic rough microsomes, we found that mitochondrial Bcl-2 inhibitor of transcription (BIT1 localized in the early secretory pathway and accumulated in the Golgi complex. Using both a chimeric protein of the luminal and transmembrane domains of ER-resident TRAPalpha and the cytosolic domain of BIT1, and silencing of BIT1 expression, we perturbed endogenous BIT1 oligomerization and localization to the Golgi. This led to enhanced ERK signaling from the Golgi complex, which resulted in improved stress resistance. This work provides the first evidence for the existence of ER microdomains that are involved in the regulation of BIT1 structure and trafficking, and identifies BIT1 as a negative regulator of the ERK-MAPK signaling pathway in the Golgi.

post to: CiteULike

Early initiation of a replication origin tethered at the nuclear periphery.

Publication Date: 2010 Mar 2 PMID: 20197407
Authors: Ebrahimi, H. - Robertson, E. D. - Taddei, A. - Gasser, S. M. - Donaldson, A. D. - Hiraga, S. I.
Journal: J Cell Sci

Peripheral nuclear localization of chromosomal loci correlates with late replication in yeast and metazoan cells. To test whether peripheral positioning can impose late replication, we examined whether artificial tethering of an early-initiating replication origin to the nuclear periphery delays its replication in budding yeast. We tested the effects of three different peripheral tethering constructs on the time of replication of the early replication origin ARS607. Using the dense-isotope transfer method to assess replication time, we found that ARS607 still replicates early when tethered to the nuclear periphery using the Yif1 protein or a fragment of Sir4, whereas tethering using a Yku80 construct produces only a very slight replication delay. Single-cell microscopic analysis revealed no correlation between peripheral positioning of ARS607 in individual cells and delayed replication. Overall, our results demonstrate that a replication origin can initiate replication early in S phase, even if artificially relocated to the nuclear periphery.

post to: CiteULike

Changes in Bni4 localization induced by cell stress in Saccharomyces cerevisiae.

Publication Date: 2010 Mar 2 PMID: 20197406
Authors: Larson, J. R. - Kozubowski, L. - Tatchell, K.
Journal: J Cell Sci

Septin complexes at the bud neck in Saccharomyces cerevisiae serve as a scaffold for proteins involved in signaling, cell cycle control, and cell wall synthesis. Many of these bind asymmetrically, associating with either the mother- or daughter-side of the neck. Septin structures are inherently apolar so the basis for the asymmetric binding remains unknown. Bni4, a regulatory subunit of yeast protein phosphatase type 1, Glc7, binds to the outside of the septin ring prior to bud formation and remains restricted to the mother-side of the bud neck after bud emergence. Bni4 is responsible for targeting Glc7 to the mother-side of the bud neck for proper deposition of the chitin ring. We show here that Bni4 localizes symmetrically, as two distinct rings on both sides of the bud neck following energy depletion or activation of cell cycle checkpoints. Our data indicate that loss of Bni4 asymmetry can occur via at least two different mechanisms. Furthermore, we show that Bni4 has a Swe1-dependent role in regulating the cell morphogenesis checkpoint in response to hydroxyurea, which suggests that the change in localization of Bni4 following checkpoint activation may help stabilize the cell cycle regulator Swe1 during cell cycle arrest.

post to: CiteULike

The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.

Publication Date: 2010 Mar 2 PMID: 20197405
Authors: Miao, J. - Li, J. - Fan, Q. - Li, X. - Li, X. - Cui, L.
Journal: J Cell Sci

Translation regulation plays an important role during gametocytogenesis in the malaria parasite, a process that is obligatory for the transmission of the parasite through mosquito vectors. In this study we determined the function of PfPuf2, a member of the Puf family of translational repressors, in gametocytogenesis of Plasmodium falciparum. Tagging of the endogenous PfPuf2 protein with green fluorescent protein showed that PfPuf2 was expressed in both male and female gametocytes, and the protein was localized in the cytoplasm of the parasite. Targeted disruption of the PfPuf2 gene did not affect asexual growth of the parasite, but promoted the formation of gametocytes and differentiation of male gametocytes. Complementation studies were performed to confirm that the resultant phenotypic changes were due to disruption of the PfPuf2 gene. Episomal expression of PfPuf2 under its cognate promoter almost restored the gametocytogenesis rate in a PfPuf2 disruptant to the level of the wild-type parasite. It also partially restored the effect of PfPuf2 disruption on male-female sex ratio. In addition, episomal overexpression of PfPuf2 under its cognate promoter but with a higher concentration of the selection drug or under the constitutive hsp86 promoter in both the PfPuf2-disruptant and wild-type 3D7 lines, further dramatically reduced gametocytogenesis rates and sex ratios. These findings suggest that in this early branch of eukaryotes the function of PfPuf2 is consistent with the ancestral function of suppressing differentiation proposed for Puf-family proteins.

post to: CiteULike

Polarity proteins and Rho GTPases cooperate to spatially organise epithelial actin-based protrusions.

Publication Date: 2010 Mar 2 PMID: 20197404
Authors: Georgiou, M. - Baum, B.
Journal: J Cell Sci

Different actin-filament-based structures co-exist in many cells. Here, we characterise dynamic actin-based protrusions that form at distinct positions within columnar epithelial cells, focusing on basal filopodia and sheet-like intermediate-level protrusions that extend between surrounding epithelial cells. Using a genetic analysis, we found that the form and distribution of these actin-filament-based structures depends on the activities of apical polarity determinants, not on basal integrin signalling. Bazooka/Par3 acts upstream of the RacGEF Sif/TIAM1 to limit filopodia to the basal domain, whereas Cdc42, aPKC and Par6 are required for normal protrusion morphology and dynamics. Downstream of these polarity regulators, Sif/TIAM1, Rac, SCAR and Arp2/3 complexes catalyse actin nucleation to generate lamellipodia and filopodia, whose form depends on the level of Rac activation. Taken together, these data reveal a role for Baz/Par3 in the establishment of an intercellular gradient of Rac inhibition, from apical to basal, and an intimate association between different apically concentrated Par proteins and Rho-family GTPases in the regulation of the distribution and structure of the polarised epithelial actin cytoskeleton.

post to: CiteULike

CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells.

Publication Date: 2010 Mar 2 PMID: 20197403
Authors: Odemis, V. - Boosmann, K. - Heinen, A. - Kury, P. - Engele, J.
Journal: J Cell Sci

The alternative SDF-1 (stromal cell derived factor-1 receptor, CXCR7, has been suggested to act as either a scavenger of extracellular SDF-1 or a modulator of the primary SDF-1 receptor, CXCR4. CXCR7, however, also directly affects the function of various tumor-cell types. Here, we demonstrate that CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. Cultured cortical astrocytes and peripheral nerve Schwann cells exhibit comparable total and cell-surface levels of expression of both SDF-1 receptors. Stimulation of astrocytes with SDF-1 resulted in the temporary activation of Erk1/2, Akt and PKCzeta/lambda, but not p38 and PKCalpha/beta. Schwann cells showed SDF-1-induced activation of Erk1/2, Akt and p38, but not PKCalpha/beta and PKCzeta/lambda. The respective signalling pattern remained fully inducible in astrocytes from CXCR4-deficient mice, but was abrogated following depletion of astrocytic CXCR7 by RNAi. In Schwann cells, RNAi-mediated depletion of either CXCR4 or CXCR7 silenced SDF-1 signalling. The findings of the astrocytic receptor-depletion experiments were reproduced by CXCR7 antagonist CCX754, but not by CXCR4 antagonist AMD3100, both of which abolished astrocytic SDF-1 signalling. Further underlining the functional importance of CXCR7 signalling in glial cells, we show that the mitogenic effects of SDF-1 on both glial cell types are impaired upon depleting CXCR7.

post to: CiteULike

Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells.

Publication Date: 2010 Mar 15 PMID: 20179105
Authors: Bystrova, M. F. - Romanov, R. A. - Rogachevskaja, O. A. - Churbanov, G. D. - Kolesnikov, S. S.
Journal: J Cell Sci

Three types of morphologically and functionally distinct taste cells operate in the mammalian taste bud. We demonstrate here the expression of two G-protein-coupled receptors from the family C, CASR and GPRC6A, in the taste tissue and identify transcripts for both receptors in type I cells, no transcripts in type II cells and only CASR transcripts in type III cells, by using the SMART-PCR RNA amplification method at the level of individual taste cells. Type I taste cells responded to calcimimetic NPS R-568, a stereoselective CASR probe, with Ca(2+) transients, whereas type I and type II cells were not specifically responsive. Consistent with these findings, certain amino acids stimulated PLC-dependent Ca(2+) signaling in type III cells, but not in type I and type II cells, showing the following order of efficacies: Phe~Glu>Arg. Thus, CASR is coupled to Ca(2+) mobilization solely in type III cells. CASR was cloned from the circumvallate papilla into a pIRES2-EGFP plasmid and heterologously expressed in HEK-293 cells. The transfection with CASR enabled HEK-293 cells to generate Ca(2+) transients in response to the amino acids, of which, Phe was most potent. This observation and some other facts favor CASR as the predominant receptor subtype endowing type III cells with the ability to detect amino acids. Altogether, our results indicate that type III cells can serve a novel chemosensory function by expressing the polymodal receptor CASR. A role for CASR and GPRC6A in physiology of taste cells of the type I remains to be unveiled.

post to: CiteULike

Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1.

Publication Date: 2010 Mar 15 PMID: 20179104
Authors: Mai, S. - Klinkenberg, M. - Auburger, G. - Bereiter-Hahn, J. - Jendrach, M.
Journal: J Cell Sci

Mitochondria display different morphologies, depending on cell type and physiological situation. In many senescent cell types, an extensive elongation of mitochondria occurs, implying that the increase of mitochondrial length in senescence could have a functional role. To test this hypothesis, human endothelial cells (HUVECs) were aged in vitro. Young HUVECs had tubular mitochondria, whereas senescent cells were characterized by long interconnected mitochondria. The change in mitochondrial morphology was caused by downregulation of the expression of Fis1 and Drp1, two proteins regulating mitochondrial fission. Targeted photodamage of mitochondria induced the formation of reactive oxygen species (ROS), which triggered mitochondrial fragmentation and loss of membrane potential in young cells, whereas senescent cells proved to be resistant. Alterations of the Fis1 and Drp1 expression levels also influenced the expression of the putative serine-threonine kinase PINK1, which is associated with the PARK6 variant of Parkinson's disease. Downregulation of PINK1 or overexpression of a PINK1 mutant (G309D) increased the sensitivity against ROS in young cells. These results indicate that there is a Drp1- and Fis1-induced, and PINK1-mediated protection mechanism in senescent cells, which, when compromised, could contribute to the age-related progression of Parkinson's disease and arteriosclerosis.

post to: CiteULike

Active Mek2 as a regulatory scaffold that promotes Pin1 binding to BPGAP1 to suppress BPGAP1-induced acute Erk activation and cell migration.

Publication Date: 2010 Mar 15 PMID: 20179103
Authors: Pan, C. Q. - Liou, Y. C. - Low, B. C.
Journal: J Cell Sci

BPGAP1 is a multidomain Rho GTPase-activating protein (RhoGAP) that promotes Erk activation and cell motility. However, the molecular mechanism of how these two processes are linked and regulated remains unclear. Here, we show that the RhoGAP domain of BPGAP1 interacts with the peptidyl-prolyl cis/trans isomerase (PPI) Pin1, leading to enhanced GAP activity towards RhoA. BPGAP1 also interacted with wild-type and constitutively active Mek2, but not with its kinase-dead mutant. However, only active Mek2 could bind Pin1, acting as a scaffold to bridge Pin1 and BPGAP1 in a manner that involves the release of an autoinhibited proline-rich motif, 186-PPLP-189, proximal to the RhoGAP domain. This allows the non-canonical 186-PPLP-189 and 256-DDYGD-260 motifs of the proline-rich region and RhoGAP domain of BPGAP1 to become accessible to concerted binding by the WW and PPI domains of Pin1, respectively. Interestingly, Pin1 knockdown led to 'super-induction' of BPGAP1-induced acute, but not chronic, Erk activation upon epidermal growth factor stimulation, in a process independent of GAP modulation. Reintroducing Pin1, but not its catalytic or non-binding mutants, reversed the effect and inhibited cell migration induced by coexpression of BPGAP1 and active Mek2. Thus, Pin1 regulates BPGAP1 function in Rho and Erk signalling, with active Mek2 serving as a novel regulatory scaffold that promotes crosstalk between RhoGAP, Pin1 and Erk in the regulation of cell migration.

post to: CiteULike

Analysis of integrin turnover in fly myotendinous junctions.

Publication Date: 2010 Mar 15 PMID: 20179102
Authors: Yuan, L. - Fairchild, M. J. - Perkins, A. D. - Tanentzapf, G.
Journal: J Cell Sci

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. We used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. We found that the IAC undergoes turnover in MTJs and that this process is mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. We propose that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

post to: CiteULike

A growth stimulus is needed for IGF-1 to induce skeletal muscle hypertrophy in vivo.

Publication Date: 2010 Mar 15 PMID: 20179101
Authors: Shavlakadze, T. - Chai, J. - Maley, K. - Cozens, G. - Grounds, G. - Winn, N. - Rosenthal, N. - Grounds, M. D.
Journal: J Cell Sci

Here, we characterise new strains of normal and dystrophic (mdx) mice that overexpress Class 2 IGF-1 Ea in skeletal myofibres. We show that transgenic mice have increased muscle levels of IGF-1 (~13-26 fold) and show striking muscle hypertrophy (~24-56% increase in mass). Adult normal muscles were resistant to elevated IGF-1; they reached adult steady state and maintained the same mass from 3 to 12 months. By contrast, dystrophic muscles from mdx/IGF-1(C2:Ea) mice continued to increase in mass during adulthood. IGF-1 signalling was evident only in muscles that were growing as a result of normal postnatal development (23-day-old mice) or regenerating in response to endogenous necrosis (adult mdx mice). Increased phosphorylation of Akt at Ser473 was not evident in fasted normal adult transgenic muscles, but was 1.9-fold higher in fasted normal young transgenic muscles compared with age-matched wild-type controls and fourfold higher in fasted adult mdx/IGF-1(C2:Ea) compared with mdx muscles. Muscles of adult mdx/IGF-1(C2:Ea) mice showed higher p70(S6K)(Thr421/Ser424) phosphorylation and both young transgenic and adult mdx/IGF-1(C2:Ea) mice had higher phosphorylation of rpS6(Ser235/236). The level of mRNA encoding myogenin was increased in normal young (but not adult) transgenic muscles, indicating enhanced myogenic differentiation. These data demonstrate that elevated IGF-1 has a hypertrophic effect on skeletal muscle only in growth situations.

post to: CiteULike

Ca2+ influx and protein scaffolding via TRPC3 sustain PKC{beta} and ERK activation in B cells.

Publication Date: 2010 Mar 15 PMID: 20179100
Authors: Numaga, T. - Nishida, M. - Kiyonaka, S. - Kato, K. - Katano, M. - Mori, E. - Kurosaki, T. - Inoue, R. - Hikida, M. - Putney, J. W. Jr - Mori, Y.
Journal: J Cell Sci

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

post to: CiteULike

Retromer-mediated direct sorting is required for proper endosomal recycling of the mammalian iron transporter DMT1.

Publication Date: 2010 Mar 1 PMID: 20164305
Authors: Tabuchi, M. - Yanatori, I. - Kawai, Y. - Kishi, F.
Journal: J Cell Sci

Endosomal recycling of the mammalian iron transporter DMT1 is assumed to be important for efficient and rapid uptake of iron across the endosomal membrane in the transferrin cycle. Here, we show that the retromer, a complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network, is required for endosomal recycling of DMT1-II, an alternative splicing isoform of DMT1. Bacterially expressed Vps26-Vsp29-Vsp35 trimer, a retromer cargo recognition complex, specifically binds to the cytoplasmic tail domain of DMT1-II in vitro. In particular, this binding is dependent on a specific hydrophobic motif of DMT1-II, which is required for its endosomal recycling. DMT1-II colocalizes with the Vps35 subunit of the retromer in TfR-positive endosomes. Depletion of the retromer by siRNA against Vps35 leads to mis-sorting of DMT1-II to LAMP2-positive structures, and expression of siRNA-resistant Vps35 can rescue this effect. These findings demonstrate that the retromer recognizes the recycling signal of DMT1-II and ensures its proper endosomal recycling.

post to: CiteULike

Functional molecular imaging of ILK-mediated Akt/PKB signaling cascades and the associated role of beta-parvin.

Publication Date: 2010 Mar 1 PMID: 20164304
Authors: Kimura, M. - Murakami, T. - Kizaka-Kondoh, S. - Itoh, M. - Yamamoto, K. - Hojo, Y. - Takano, M. - Kario, K. - Shimada, K. - Kobayashi, E.
Journal: J Cell Sci

Visualization and quantification of the dynamics of protein-protein interactions in living cells can be used to explore the macromolecular events involved in signal transduction processes. In this study, functional molecular imaging using a luciferase-based complementation method demonstrated how the integrin-linked kinase (ILK)-mediated protein complex controls downstream signals. The luciferase complementation assay showed that Akt1 preferentially binds to beta-parvin rather than to ILK within the complex. Moreover, photon flux from the interaction between beta-parvin and Akt1 increased following serum stimulation, and the beta-parvin-Akt1 interaction was dependent on phosphoinositide 3-kinase. Intriguingly, small interfering (si)RNA-mediated beta-parvin knockdown increased photon flux from the interaction between ILK and Akt1, leading to stabilization of hypoxia-inducible factor-1alpha and increased expression of vascular endothelial growth factor-A. These data from functional molecular imaging demonstrated that beta-parvin plays a regulatory role in the ILK-mediated Akt (also called protein kinase B) signaling cascades, suggesting that beta-parvin might be a crucial modulator of cell survival.

post to: CiteULike

Roadblocks en route to the clinical application of induced pluripotent stem cells.

Publication Date: 2010 Mar 1 PMID: 20164303
Authors: Lowry, W. E. - Quan, W. L.
Journal: J Cell Sci

Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.

post to: CiteULike

The catenin family at a glance.

Publication Date: 2010 Mar 1 PMID: 20164302
Authors: McCrea, P. D. - Gu, D.
Journal: J Cell Sci

post to: CiteULike

GSK-3{beta} promotes cell survival by modulating Bif-1-dependent autophagy and cell death.

Publication Date: 2010 Mar 15 PMID: 20159967
Authors: Yang, J. - Takahashi, Y. - Cheng, E. - Liu, J. - Terranova, P. F. - Zhao, B. - Thrasher, J. B. - Wang, H. G. - Li, B.
Journal: J Cell Sci

Glycogen synthase kinase 3 beta (GSK-3beta) is constantly active in cells and its activity increases after serum deprivation, indicating that GSK-3beta might play a major role in cell survival under serum starvation. In this study, we attempted to determine how GSK-3beta promotes cell survival after serum depletion. Under full culture conditions (10% FBS), GSK-3beta inhibition with chemical inhibitors or siRNAs failed to induce cell death in human prostate cancer cells. By contrast, under conditions of serum starvation, a profound necrotic cell death was observed as evidenced by cellular morphologic features and biochemical markers. Further analysis revealed that GSK-3beta-inhibition-induced cell death was in parallel with an extensive autophagic response. Interestingly, blocking the autophagic response switched GSK-3beta-inhibition-induced necrosis to apoptotic cell death. Finally, GSK-3beta inhibition resulted in a remarkable elevation of Bif-1 protein levels, and silencing Bif-1 expression abrogated GSK-3beta-inhibition-induced autophagic response and cell death. Taken together, our study suggests that GSK-3beta promotes cell survival by modulating Bif-1-dependent autophagic response and cell death.

post to: CiteULike

Sirtuin-3 deacetylation of cyclophilin D induces dissociation of hexokinase II from the mitochondria.

Publication Date: 2010 Mar 15 PMID: 20159966
Authors: Shulga, N. - Wilson-Smith, R. - Pastorino, J. G.
Journal: J Cell Sci

We demonstrate that the transition from a reliance on glycolysis to oxidative phosphorylation in a transformed cell line is dependent on an increase in the levels and activity of sirtuin-3. Sirtuin-3 deacetylates cyclophilin D, diminishing its peptidyl-prolyl cis-trans isomerase activity and inducing its dissociation from the adenine nucleotide translocator. Moreover, the sirtuin-3-induced inactivation of cyclophilin D causes a detachment of hexokinase II from the mitochondria that is necessary for stimulation of oxidative phosphorylation. These results might have important implications for the role of sirtuin-3 in the metabolism of some cancer cells and their susceptibility to mitochondrial injury and cytotoxicity.

post to: CiteULike

Human dermal stem cells differentiate into functional epidermal melanocytes.

Publication Date: 2010 Mar 15 PMID: 20159965
Authors: Li, L. - Fukunaga-Kalabis, M. - Yu, H. - Xu, X. - Kong, J. - Lee, J. T. - Herlyn, M.
Journal: J Cell Sci

Melanocytes sustain a lifelong proliferative potential, but a stem cell reservoir in glabrous skin has not yet been found. Here, we show that multipotent dermal stem cells isolated from human foreskins lacking hair follicles are able to home to the epidermis to differentiate into melanocytes. These dermal stem cells, grown as three-dimensional spheres, displayed a capacity for self-renewal and expressed NGFRp75, nestin and OCT4, but not melanocyte markers. In addition, cells derived from single-cell clones were able to differentiate into multiple lineages including melanocytes. In a three-dimensional skin equivalent model, sphere-forming cells differentiated into HMB45-positive melanocytes, which migrated from the dermis to the epidermis and aligned singly among the basal layer keratinocytes in a similar fashion to pigmented melanocytes isolated from the epidermis. The dermal stem cells were negative for E-cadherin and N-cadherin, whereas they acquired E-cadherin expression and lost NGFRp75 expression upon contact with epidermal keratinocytes. These results demonstrate that stem cells in the dermis of human skin with neural-crest-like characteristics can become mature epidermal melanocytes. This finding could significantly change our understanding of the etiological factors in melanocyte transformation and pigmentation disorders; specifically, that early epigenetic or genetic alterations leading to transformation may take place in the dermis rather than in the epidermis.

post to: CiteULike

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.

Publication Date: 2010 Mar 15 PMID: 20159964
Authors: Silverman, J. M. - Clos, J. - De'oliveira, C. C. - Shirvani, O. - Fang, Y. - Wang, C. - Foster, L. J. - Reiner, N. E.
Journal: J Cell Sci

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

post to: CiteULike

Dual regulation of a Dictyostelium STAT by cGMP and Ca2+ signalling.

Publication Date: 2010 Mar 15 PMID: 20159963
Authors: Araki, T. - van Egmond, W. N. - van Haastert, P. J. - Williams, J. G.
Journal: J Cell Sci

When cells are exposed to hyperosmotic stress, the Dictyostelium STAT orthologue STATc is rapidly tyrosine phosphorylated. Previous observations suggest a non-paradigmatic mode of STAT activation, whereby stress-induced serine phosphorylation of the PTP3 protein tyrosine phosphatase inhibits its activity towards STATc. We show that two serine residues in PTP3, S448 and S747, are rapidly phosphorylated after osmotic stress. cGMP is a second messenger for hyperosmotic stress response and 8-bromo-cGMP, a membrane-permeable form of cGMP, is a known activator of STATc. GbpC, a cGMP-binding Ras guanine nucleotide exchange factor protein, is a founder member of a protein family that includes LRRK2, the gene commonly mutated in familial Parkinson's disease. Genetic ablation of gbpC prevents STATc activation by 8-bromo-cGMP. However, osmotic-stress-induced activation of STATc occurs normally in the gbpC null mutant. Moreover, 8-bromo-cGMP does not stimulate phosphorylation of S448 and S747 of PTP3 in a wild-type strain. These facts imply the occurrence of redundant activation pathways. We present evidence that intracellular Ca(2+) is a parallel second messenger, by showing that agents that elevate intracellular Ca(2+) levels are potent STATc activators that stimulate phosphorylation of S448 and S747. We propose that stress-induced cGMP signalling exerts its stimulatory effect by potentiating the activity of a semi-constitutive tyrosine kinase that phosphorylates STATc, whereas parallel, stress-induced Ca(2+) signalling represses STATc dephosphorylation through its inhibitory effect on PTP3.

post to: CiteULike

Opposing effects of retinoic acid and FGF9 on Nanos2 expression and meiotic entry of mouse germ cells.

Publication Date: 2010 Mar 15 PMID: 20159962
Authors: Barrios, F. - Filipponi, D. - Pellegrini, M. - Paronetto, M. P. - Di Siena, S. - Geremia, R. - Rossi, P. - De Felici, M. - Jannini, E. A. - Dolci, S.
Journal: J Cell Sci

In the mouse, three genes that are homologous to the Drosophila Nanos (Nos) gene have been identified. Deletion of one of these genes, Nanos2, results in male sterility, owing to loss of germ cells during fetal life. Before apoptosis, Nanos2-null gonocytes enter meiosis, suggesting that Nanos2 functions as a meiotic repressor. Here, we show that Nanos2 is continuously expressed in male germ cells from fetal gonocytes to postnatal spermatogonial stem cells. We observed that the promeiotic factor AtRA, an analog of retinoic acid (RA), downregulates NANOS2 levels, in both fetal and postnatal gonocytes, while promoting meiosis. Interestingly, FGF9, a growth factor crucial for sex differentiation and survival of fetal gonocytes, upregulates levels of NANOS2 in both male and female primordial germ cells (PGCs) and in premeiotic spermatogonia. This effect was paralleled by an impairment of meiotic entry, suggesting that FGF9 acts as an inhibitor of meiosis through the upregulation of Nanos2. We found that NANOS2 interacts with PUM2, and that these two proteins colocalize in the ribonucleoparticle and polysomal fractions on sucrose gradients, supporting the notion that they bind RNA. Finally, we found that recombinant NANOS2 binds to two spermatogonial mRNAs, Gata2 and Taf7l, which are involved in germ-cell differentiation.

post to: CiteULike

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

Publication Date: 2010 Mar 15 PMID: 20159961
Authors: Giachello, C. N. - Fiumara, F. - Giacomini, C. - Corradi, A. - Milanese, C. - Ghirardi, M. - Benfenati, F. - Montarolo, P. G.
Journal: J Cell Sci

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.

post to: CiteULike

Drosophila Rel proteins are central regulators of a robust, multi-organ immune network.

Publication Date: 2010 Feb 15 PMID: 20145002
Authors: Matova, N. - Anderson, K. V.
Journal: J Cell Sci

Survival of all animals depends on effective protection against infection. In Drosophila, opportunistic infection kills larvae if they lack the Rel/NF-kappaB proteins Dorsal and Dif. We have used tissue-specific expression of Dif and Dorsal to reveal that these Rel proteins act in three different tissues to defend larvae from infection. Dif and Dorsal act in circulating blood cells, where they are required autonomously to promote blood-cell survival and phagocytosis of microorganisms. We show that a major transcriptional target of Dorsal and Dif in blood cells is Drosophila IAP1, a gene protecting these cells from death. We find that in addition to their autonomous role in blood-cell survival, Dif and Dorsal also act in the fat body to produce factors that promote blood-cell viability. These Rel proteins act in the epidermis to prevent infection by maintaining a barrier to microbial entry. Dorsal or Dif in any one of the three tissues is sufficient to defend the animal from opportunistic infection. Thus Drosophila has a multi-pronged system of defense and each branch of this network requires Rel proteins. Based on similarities between Drosophila and mammals, we propose that a Rel-dependent network is an ancient and robust framework of animal immune systems.

post to: CiteULike

Molecular mechanisms of protein and lipid targeting to ciliary membranes.

Publication Date: 2010 Feb 15 PMID: 20145001
Authors: Emmer, B. T. - Maric, D. - Engman, D. M.
Journal: J Cell Sci

Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle.

post to: CiteULike

Flagellar and ciliary beating: the proven and the possible.

Publication Date: 2010 Feb 15 PMID: 20145000
Authors: Lindemann, C. B. - Lesich, K. A.
Journal: J Cell Sci

The working mechanism of the eukaryotic flagellar axoneme remains one of nature's most enduring puzzles. The basic mechanical operation of the axoneme is now a story that is fairly complete; however, the mechanism for coordinating the action of the dynein motor proteins to produce beating is still controversial. Although a full grasp of the dynein switching mechanism remains elusive, recent experimental reports provide new insights that might finally disclose the secrets of the beating mechanism: the special role of the inner dynein arms, especially dynein I1 and the dynein regulatory complex, the importance of the dynein microtubule-binding affinity at the stalk, and the role of bending in the selection of the active dynein group have all been implicated by major new evidence. This Commentary considers this new evidence in the context of various hypotheses of how axonemal dynein coordination might work.

post to: CiteULike

The perennial organelle: assembly and disassembly of the primary cilium.

Publication Date: 2010 Feb 15 PMID: 20144999
Authors: Seeley, E. S. - Nachury, M. V.
Journal: J Cell Sci

Primary cilia contain signaling receptors of diverse classes, and ciliary dysfunction results in a variety of developmental defects. Thus, primary cilia are thought to have an important role in sensing and transducing cellular signals. Although there is clear evidence demonstrating that these organelles are assembled and disassembled dynamically as cells progress through the cell cycle, the mechanisms by which the cell cycle controls the assembly and disassembly of the primary cilium remain poorly understood. In this Commentary, we review the basic cellular mechanisms that underlie the early stages of cilium assembly and discuss how the cell cycle communicates with the ciliation program. A commonly held view is that ciliation occurs exclusively in cells that have exited the cell cycle and entered quiescence or differentiation. However, this concept is at odds with the finding that, during development, many actively proliferating cells require cilia-mediated signaling pathways to instruct their developmental fate. Here, we reassess the quiescence-centric view of ciliation by reviewing historic and current literature. We discuss ample evidence that cilia are in fact present on many proliferating cells, and that a transient peak of ciliation before the G1-S transition might be tightly coupled to entry into the DNA replication phase. Finally, we touch on the relationship between the ciliation and cell-division cycles and the tissue distribution of primary cilia in order to highlight potential roles for the primary cilium in restraining cells from the hyperproliferative state that contributes to cancer.

post to: CiteULike

Sensory reception is an attribute of both primary cilia and motile cilia.

Publication Date: 2010 Feb 15 PMID: 20144998
Authors: Bloodgood, R. A.
Journal: J Cell Sci

A recent cluster of papers has shown that motile cilia in the respiratory and reproductive tracts of humans and other mammals can exhibit sensory functions, a function previously attributed primarily to non-motile primary cilia. This leads to a new paradigm that all cilia and flagella (both motile and primary) can mediate sensory functions. However, examination of the literature shows that evidence of sensory functions of motile cilia and flagella is widespread in studies of invertebrates, and extends as back as far as 1899. In this Opinion article, I review the recent and historical findings that motile cilia have a variety of sensory functions, and discuss how this concept has in fact been evolving for the past century.

post to: CiteULike

The primary cilium at a glance.

Publication Date: 2010 Feb 15 PMID: 20144997
Authors: Satir, P. - Pedersen, L. B. - Christensen, S. T.
Journal: J Cell Sci

post to: CiteULike

Integrins stimulate E-cadherin-mediated intercellular adhesion by regulating Src-kinase activation and actomyosin contractility.

Publication Date: 2010 Mar 1 PMID: 20144995
Authors: Martinez-Rico, C. - Pincet, F. - Thiery, J. P. - Dufour, S.
Journal: J Cell Sci

Cadherins and integrins are major adhesion molecules regulating cell-cell and cell-matrix interactions. In vitro and in vivo studies have demonstrated the existence of crosstalk between integrins and cadherins in cell adhesion and motility. We used a dual pipette assay to measure the force required to separate E-cadherin-producing cell doublets and to investigate the role of integrin in regulating the strength of intercellular adhesion. A greater force was required to separate cell doublets bound to fibronectin or vitronectin-coated beads than for doublets bound to polylysine-coated beads. This effect depended on cell spreading and the duration of stimulation. Cells expressing type II cadherin-7 also responded to fibronectin stimulation to produce a higher intercellular adhesion. Establishment of cadherin-mediated adhesion needed ROCK, MLCK and myosin ATPase II activity. The regulation of intercellular adhesion strength by integrin stimulation required activation of Src family kinases, ROCK and actomyosin contractility. These findings highlight the importance and mechanisms of molecular crosstalk between cadherins and integrins in the control of cell plasticity during histogenesis and morphogenesis.

post to: CiteULike

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length.

Publication Date: 2010 Mar 1 PMID: 20144994
Authors: Savoian, M. S. - Glover, D. M.
Journal: J Cell Sci

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length.

post to: CiteULike

Control of mitotic and meiotic centriole duplication by the Plk4-related kinase ZYG-1.

Publication Date: 2010 Mar 1 PMID: 20144993
Authors: Peters, N. - Perez, D. E. - Song, M. H. - Liu, Y. - Muller-Reichert, T. - Caron, C. - Kemphues, K. J. - O'Connell, K. F.
Journal: J Cell Sci

Centriole duplication is of crucial importance during both mitotic and male meiotic divisions, but it is currently not known whether this process is regulated differently during the two modes of division. In Caenorhabditis elegans, the kinase ZYG-1 plays an essential role in both mitotic and meiotic centriole duplication. We have found that the C-terminus of ZYG-1 is necessary and sufficient for targeting to centrosomes and is important for differentiating mitotic and meiotic centriole duplication. Small truncations of the C-terminus dramatically lower the level of ZYG-1 at mitotic centrosomes but have little effect on the level of ZYG-1 at meiotic centrosomes. Interestingly, truncation of ZYG-1 blocks centrosome duplication in the mitotic cycle but leads to centrosome amplification in the meiotic cycle. Meiotic centriole amplification appears to result from the overduplication of centrioles during meiosis I and leads to the formation of multipolar meiosis II spindles. The extra centrioles also disrupt spermatogenesis by inducing the formation of supernumerary fertilization-competent spermatids that contain abnormal numbers of chromosomes and centrioles. Our data reveal differences in the regulation of mitotic and meiotic centrosome duplication, particularly with regard to ZYG-1 activity, and reveal an important role for centrosomes in spermatid formation.

post to: CiteULike

Role of TI-VAMP and CD82 in EGFR cell-surface dynamics and signaling.

Publication Date: 2010 Mar 1 PMID: 20144992
Authors: Danglot, L. - Chaineau, M. - Dahan, M. - Gendron, M. C. - Boggetto, N. - Perez, F. - Galli, T.
Journal: J Cell Sci

The v-SNARE TI-VAMP (VAMP7) mediates exocytosis during neuritogenesis, phagocytosis and lysosomal secretion. It localizes to endosomes and lysosomes but also to the trans-Golgi network. Here we show that depletion of TI-VAMP enhances the endocytosis of activated EGF receptor (EGFR) without affecting constitutive endocytosis of EGFR, or transferrin uptake. This increased EGFR internalization is mainly clathrin dependent. Searching for defects in EGFR regulators, we found that TI-VAMP depletion reduces the cell surface amount of CD82, a tetraspanin known to control EGFR localization in microdomains. We further show that TI-VAMP is required for secretion from the Golgi apparatus to the cell surface, and that TI-VAMP-positive vesicles transport CD82. Quantum dots video-microscopy indicates that depletion of TI-VAMP, or its cargo CD82, restrains EGFR diffusion and the area explored by EGFR at the cell surface. Both depletions also impair MAPK signaling and enhance endocytosis of activated EGFR by increased recruitment of AP-2. These results highlight the role of TI-VAMP in the secretory pathway of a tetraspanin, and support a model in which CD82 allows EGFR entry in microdomains that control its clathrin-dependent endocytosis and signaling.

post to: CiteULike

Regulated increase in folding capacity prevents unfolded protein stress in the ER.

Publication Date: 2010 Mar 1 PMID: 20144991
Authors: Christis, C. - Fullaondo, A. - Schildknegt, D. - Mkrtchian, S. - Heck, A. J. - Braakman, I.
Journal: J Cell Sci

Stimulation of thyrocytes with thyroid stimulating hormone (TSH) leads to a morphological change and a massive increase in thyroglobulin (Tg) production. Although Tg is a demanding client of the endoplasmic reticulum (ER), its increase did not result in significant accumulation of unfolded protein in the ER. Instead, ER chaperones and folding enzymes reached maximum synthesis rates immediately after TSH stimulation, before significant upregulation of Tg synthesis. The resulting increase in folding capacity before client protein production prevented cellular unfolded-protein stress, confirmed by the silence of the most conserved branch of the unfolded protein response. Thyrocytes set an example of physiological adaptation of cells to a future potentially stress-causing situation, which suggests a general strategy for both non-secretory and specialized secretory cells.

post to: CiteULike

Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin.

Publication Date: 2010 Mar 1 PMID: 20144990
Authors: Nakashima, A. - Sato, T. - Tamanoi, F.
Journal: J Cell Sci

Cellular activities are regulated by environmental stimuli through protein phosphorylation. Target of rapamycin (TOR), a serine/threonine kinase, plays pivotal roles in cell proliferation and cell growth in response to nutrient status. In Schizosaccharomyces pombe, TORC1, which contains Tor2, plays crucial roles in nutrient response. Here we find a nitrogen-regulated phosphoprotein, p27, in S. pombe using the phospho-Akt substrate antibody. Response of p27 phosphorylation to nitrogen availability is mediated by TORC1 and the TSC-Rhb1 signaling, but not by TORC2 or other nutrient stress-related pathways. Database and biochemical analyses indicate that p27 is identical to ribosomal protein S6 (Rps6). Ser235 and Ser236 in Rps6 are necessary for Rps6 phosphorylation by TORC1. These Rps6 phosphorylations are dispensable for cell viability. Rps6 phosphorylation by TORC1 also responds to availability of glucose and is inhibited by osmotic and oxidative stresses. Rapamycin inhibits the ability of TORC1 to phosphorylate Rps6, owing to interaction of the rapamycin-FKBP12 complex with the FRB domain in Tor2. Rapamycin also leads to a decrease in cell size in a TORC1-dependent manner. Our findings demonstrate that the nutrient-responsive and rapamycin-sensitive TORC1-S6 signaling exists in S. pombe, and that this pathway plays a role in cell size control.

post to: CiteULike

Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division.

Publication Date: 2010 Mar 1 PMID: 20144989
Authors: Wang, L. H. - Mayer, B. - Stemmann, O. - Nigg, E. A.
Journal: J Cell Sci

Sister chromatid cohesion is mediated by DNA catenation and proteinaceous cohesin complexes. The recent visualization of PICH (Plk1-interacting checkpoint helicase)-coated DNA threads in anaphase cells raises new questions as to the role of DNA catenation and its regulation in time and space. In the present study we show that persistent DNA catenation induced by inhibition of Topoisomerase-IIalpha can contribute to sister chromatid cohesion in the absence of cohesin complexes and that resolution of catenation is essential for abscission. Furthermore, we use an in vitro chromatid separation assay to investigate the temporal and functional relationship between cohesin removal and Topoisomerase-IIalpha-mediated decatenation. Our data suggest that centromere decatenation can occur only after separase activation and cohesin removal, providing a plausible explanation for the persistence of centromere threads after anaphase onset.

post to: CiteULike

Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP.

Publication Date: 2010 Mar 1 PMID: 20144988
Authors: Dikovskaya, D. - Li, Z. - Newton, I. P. - Davidson, I. - Hutchins, J. R. - Kalab, P. - Clarke, P. R. - Nathke, I. S.
Journal: J Cell Sci

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-beta as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of Apc, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

post to: CiteULike

Osteoblastic molecular scaffold Gab1 is required for maintaining bone homeostasis.

Publication Date: 2010 Mar 1 PMID: 20124419
Authors: Weng, T. - Mao, F. - Wang, Y. - Sun, Q. - Li, R. - Yang, G. - Zhang, X. - Luo, J. - Feng, G. S. - Yang, X.
Journal: J Cell Sci

The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, plays a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. However, its biological role in osteoblast activity and postnatal bone metabolism remains unclear. To elucidate the in vivo function of Gab1 in postnatal bone remodeling, we generated osteoblast-specific Gab1 knockout mice. Disruption of Gab1 expression in osteoblasts led to decreased trabecular bone mass with a reduced bone formation rate and a decreased bone resorption. Bones from Gab1 mutants also exhibited inferior mechanical properties. Moreover, primary osteoblasts from Gab1 mutant mice demonstrated markedly suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased expression of receptor activator of NF-kappaB ligand (RANKL). Activation of serine-threonine Akt kinase and extracellular signal-regulated kinase in response to insulin and insulin-like growth factor 1 was attenuated in Gab1 mutant osteoblasts. Our results show that Gab1-mediated signals in osteoblasts are crucial for normal postnatal bone homeostasis.

post to: CiteULike

Sgo1 establishes the centromeric cohesion protection mechanism in G2 before subsequent Bub1-dependent recruitment in mitosis.

Publication Date: 2010 Mar 1 PMID: 20124418
Authors: Perera, D. - Taylor, S. S.
Journal: J Cell Sci

Bub1 was one of the first protein kinases identified as a component of the spindle-assembly checkpoint, a surveillance mechanism that delays anaphase onset until all chromosomes are stably attached to spindle microtubules. Whereas the kinase activity of Bub1 is not required for checkpoint function in yeast, its requirement in mammalian cells is still unclear. Using a complementation assay with bona fide BUB1-null mouse embryonic fibroblasts, we show that the kinase activity of Bub1 is not required for checkpoint function or chromosome alignment. Its activity is, however, required for centromeric localisation of Sgo1, a known protector of centromeric cohesion. Despite the absence of Sgo1 from mitotic centromeres in cells devoid of Bub1 activity, centromeric cohesion is still maintained until anaphase. An explanation for this comes from observations showing that Sgo1 is first recruited to centromeric heterochromatin in G2, but then becomes diffusely localised throughout the nucleus in early prophase, before returning to centromeres later in prophase. Importantly, whereas centromeric localisation of Sgo1 in prophase is dependent on the kinase activity of Bub1, its recruitment to centromeric heterochromatin in G2 is not. Rather, the localisation of Sgo1 in G2 is abolished when heterochromatin protein 1 is not bound to centromeric heterochromatin. Thus, it seems that Sgo1 sets up the centromeric protection mechanism in G2, but that its Bub1-dependent localisation to centromeres during mitosis is not required to maintain cohesion.

post to: CiteULike

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion.

Publication Date: 2010 Mar 1 PMID: 20124417
Authors: Leman, A. R. - Noguchi, C. - Lee, C. Y. - Noguchi, E.
Journal: J Cell Sci

The Timeless-Tipin protein complex has been reported to be important for replication checkpoint and normal DNA replication processes. However, the precise mechanisms by which Timeless-Tipin preserves genomic integrity are largely unclear. Here, we describe the roles of Timeless-Tipin in replication fork stabilization and sister chromatid cohesion. We show in human cells that Timeless is recruited to replication origin regions and dissociate from them as replication proceeds. Cdc45, which is known to be required for replication fork progression, shows similar patterns of origin association to those of Timeless. Depletion of Timeless-Tipin causes chromosome fragmentation and defects in damage repair in response to fork collapse, suggesting that it is required for replication fork maintenance under stress. We also demonstrate that depletion of Timeless-Tipin impairs sister chromatid cohesion and causes a defect in mitotic progression. Consistently, Timeless-Tipin co-purifies with cohesin subunits and is required for their stable association with chromatin during S phase. Timeless associates with the cohesion-promoting DNA helicase ChlR1, which, when overexpressed, partially alleviates the cohesion defect of cells depleted of Timeless-Tipin. These results suggest that Timeless-Tipin functions as a replication fork stabilizer that couples DNA replication with sister chromatid cohesion established at replication forks.

post to: CiteULike

An RbAp48-like gene regulates adult stem cells in planarians.

Publication Date: 2010 Mar 1 PMID: 20124416
Authors: Bonuccelli, L. - Rossi, L. - Lena, A. - Scarcelli, V. - Rainaldi, G. - Evangelista, M. - Iacopetti, P. - Gremigni, V. - Salvetti, A.
Journal: J Cell Sci

Retinoblastoma-associated proteins 46 and 48 (RbAp46 and RbAp48) are factors that are components of different chromatin-modelling complexes, such as polycomb repressive complex 2, the activity of which is related to epigenetic gene regulation in stem cells. To date, no direct findings are available on the in vivo role of RbAp48 in stem-cell biology. We recently identified DjRbAp48 - a planarian (Dugesia japonica) homologue of human RBAP48 - expression of which is restricted to the neoblasts, the adult stem cells of planarians. In vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction in neoblast number, we were always able to detect a significant number of these cells in DjRbAp48 RNAi animals. Parallel to the decrease in neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were detectable in RNAi animals. These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of differentiation of stem-cell progeny. We discuss our data, taking into account the possibility that DjRbAp48 might control the expression of genes necessary for cell differentiation by influencing chromatin architecture.

post to: CiteULike

Phosphorylation of adhesion- and growth-regulatory human galectin-3 leads to the induction of axonal branching by local membrane L1 and ERM redistribution.

Publication Date: 2010 Mar 1 PMID: 20124415
Authors: Diez-Revuelta, N. - Velasco, S. - Andre, S. - Kaltner, H. - Kubler, D. - Gabius, H. J. - Abad-Rodriguez, J.
Journal: J Cell Sci

Serine phosphorylation of the beta-galactoside-binding protein galectin-3 (Gal-3) impacts nuclear localization but has unknown consequences for extracellular activities. Herein, we reveal that the phosphorylated form of galectin-3 (pGal-3), adsorbed to substratum surfaces or to heparan sulphate proteoglycans, is instrumental in promoting axon branching in cultured hippocampal neurons by local actin destabilization. pGal-3 interacts with neural cell adhesion molecule L1, and enhances L1 association with Thy-1-rich membrane microdomains. Concomitantly, membrane-actin linker proteins ezrin-radixin-moesin (ERM) are recruited to the same membrane site via interaction with the intracellular domain of L1. We propose that the local regulation of the L1-ERM-actin pathway, at the level of the plasma membrane, underlies pGal-3-induced axon branching, and that galectin phosphorylation in situ could act as a molecular switch for the axon response to Gal-3.

post to: CiteULike

The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes.

Publication Date: 2010 Mar 1 PMID: 20124414
Authors: Kramer, S. - Queiroz, R. - Ellis, L. - Hoheisel, J. D. - Clayton, C. - Carrington, M.
Journal: J Cell Sci

In trypanosomes, the predominant mechanisms of regulation of gene expression are post-transcriptional. The DEAD-box RNA helicase DHH1 was identified in a screen for gene products that are necessary for the instability of the GPI-PLC mRNA in insect-stage trypanosomes. Expression of an ATPase-deficient dhh1 mutant caused a rapid growth arrest associated with a decrease in polysomes, an increase in P-bodies and a slight decrease in average mRNA levels. However, the effect of dhh1 mutant expression on both turnover and translational repression of mRNAs was selective. Whereas there was little effect on the stability of constitutive mRNAs, the control of a large cohort of developmentally regulated mRNAs was reversed; many mRNAs normally downregulated in insect-stage trypanosomes were stabilized and many mRNAs normally upregulated decreased in level. One stabilised mRNA, ISG75, was characterised further. Despite the overall decrease in polysomes, the proportion of the ISG75 mRNA in polysomes was unchanged and the result was ISG75 protein accumulation. Our data show that specific mRNAs can escape DHH1-mediated translational repression. In trypanosomes, DHH1 has a selective role in determining the levels of developmentally regulated mRNAs.

post to: CiteULike

A design principle underlying the synchronization of oscillations in cellular systems.

Publication Date: 2010 Feb 15 PMID: 20103537
Authors: Kim, J. R. - Shin, D. - Jung, S. H. - Heslop-Harrison, P. - Cho, K. H.
Journal: J Cell Sci

Biological oscillations are found ubiquitously in cells and are widely variable, with periods varying from milliseconds to months, and scales involving subcellular components to large groups of organisms. Interestingly, independent oscillators from different cells often show synchronization that is not the consequence of an external regulator. What is the underlying design principle of such synchronized oscillations, and can modeling show that the complex consequences arise from simple molecular or other interactions between oscillators? When biological oscillators are coupled with each other, we found that synchronization is induced when they are connected together through a positive feedback loop. Increasing the coupling strength of two independent oscillators shows a threshold beyond which synchronization occurs within a few cycles, and a second threshold where oscillation stops. The positive feedback loop can be composed of either double-positive (PP) or double-negative (NN) interactions between a node of each of the two oscillating networks. The different coupling structures have contrasting characteristics. In particular, PP coupling is advantageous with respect to stability of period and amplitude, when local oscillators are coupled with a short time delay, whereas NN coupling is advantageous for a long time delay. In addition, PP coupling results in more robust synchronized oscillations with respect to amplitude excursions but not period, with applied noise disturbances compared to NN coupling. However, PP coupling can induce a large fluctuation in the amplitude and period of the resulting synchronized oscillation depending on the coupling strength, whereas NN coupling ensures almost constant amplitude and period irrespective of the coupling strength. Intriguingly, we have also observed that artificial evolution of random digital oscillator circuits also follows this design principle. We conclude that a different coupling strategy might have been selected according to different evolutionary requirements.

post to: CiteULike

Epithelial cell shape is regulated by Lulu proteins via myosin-II.

Publication Date: 2010 Feb 15 PMID: 20103536
Authors: Nakajima, H. - Tanoue, T.
Journal: J Cell Sci

Cell-shape change in epithelial structures is fundamental to animal morphogenesis. Recent studies identified myosin-II as the major generator of driving forces for cell-shape changes during morphogenesis. Lulu (Epb41l5) is a major regulator of morphogenesis, although the downstream molecular and cellular mechanisms remain obscure in mammals. In Drosophila and zebrafish, Lulu proteins were reported to negatively regulate Crumbs, an apical domain regulator, thus regulating morphogenesis. In this study, we show that mammalian Lulu activates myosin-II, thus regulating epithelial cell shape. In our experiments, Lulu expression in epithelial cells resulted in apical constriction and lateral elongation in the cells, accompanied by upregulation of myosin-II. The inhibition of myosin-II activity almost completely blocked this Lulu-driven cell-shape change. We further found that Rock participates in the myosin-II activation. Additionally, RNAi-mediated depletion of Lulu in epithelial cells resulted in disorganization of myosin-II and a concomitant loss of proper lateral domain organization in the cells. From these results, we propose that Lulu regulates epithelial cell shape by controlling myosin-II activity.

post to: CiteULike

RNAase-III enzyme Dicer maintains signaling pathways for differentiation and survival in mouse cortical neural stem cells.

Publication Date: 2010 Feb 15 PMID: 20103535
Authors: Kawase-Koga, Y. - Low, R. - Otaegi, G. - Pollock, A. - Deng, H. - Eisenhaber, F. - Maurer-Stroh, S. - Sun, T.
Journal: J Cell Sci

An important function of the RNAase-III enzyme Dicer is to process microRNA precursors into ~22-nucleotide non-coding small RNAs. But little is known about the role of Dicer in mammalian brain formation and neural stem cell (NSC) development. Here we show that Dicer plays a crucial role in controlling mouse cortical NSC development. We found that Dicer function is essential for expanding cortical neural progenitors and NSCs. We have identified a population of Dicer-deficient NSCs that can self-renew, and that display normal karyotype and heterochromatin protein expression levels but show enlarged nuclei. Dicer-deficient NSCs display abnormal differentiation and undergo cell death when mitogens are withdrawn. Dicer deletion affects the levels of many proteins, as revealed by a mass spectrometry proteomic approach. We have found that an increase of anti-survival and/or pro-apoptosis proteins and a decrease of pro-survival and/or anti-apoptosis proteins contribute to the cell death of Dicer-deficient NSCs, implying a general role for Dicer in protecting cells from apoptosis. Our results demonstrate important functions for Dicer in regulating NSC development by maintaining proper signaling pathways related to cell survival and differentiation.

post to: CiteULike

Cholesterol reduction impairs exocytosis of synaptic vesicles.

Publication Date: 2010 Feb 15 PMID: 20103534
Authors: Linetti, A. - Fratangeli, A. - Taverna, E. - Valnegri, P. - Francolini, M. - Cappello, V. - Matteoli, M. - Passafaro, M. - Rosa, P.
Journal: J Cell Sci

Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.

post to: CiteULike

Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1.

Publication Date: 2010 Feb 15 PMID: 20103533
Authors: Park, Y. Y. - Lee, S. - Karbowski, M. - Neutzner, A. - Youle, R. J. - Cho, H.
Journal: J Cell Sci

Mitochondria constantly divide and combine through fission and fusion activities. MARCH5, a mitochondrial E3 ubiquitin ligase, has been identified as a molecule that binds mitochondrial fission 1 protein (hFis1), dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2), key proteins in the control of mitochondrial fission and fusion. However, how these interactions control mitochondrial dynamics, and cellular function has remained obscure. Here, we show that shRNA-mediated MARCH5 knockdown promoted the accumulation of highly interconnected and elongated mitochondria. Cells transfected with MARCH5 shRNA or a MARCH5 RING domain mutant displayed cellular enlargement and flattening accompanied by increased senescence-associated beta-galactosidase (SA-beta-Gal) activity, indicating that these cells had undergone cellular senescence. Notably, a significant increase in Mfn1 level, but not Mfn2, Drp1 or hFis1 levels, was observed in MARCH5-depleted cells, indicating that Mfn1 is a major ubiquitylation substrate. Introduction of Mfn1(T109A), a GTPase-deficient mutant form of Mfn1, into MARCH5-RNAi cells not only disrupted mitochondrial elongation, but also abolished the increase in SA-beta-Gal activity. Moreover, the aberrant mitochondrial phenotypes in MARCH5-RNAi cells were reversed by ectopic expression of Drp1, but not by hFis1, and reversion of the mitochondria morphology in MARCH5-depleted cells was accompanied by a reduction in SA-beta-Gal activity. Collectively, our data indicate that the lack of MARCH5 results in mitochondrial elongation, which promotes cellular senescence by blocking Drp1 activity and/or promoting accumulation of Mfn1 at the mitochondria.

post to: CiteULike

Degradation of an intramitochondrial protein by the cytosolic proteasome.

Publication Date: 2010 Feb 15 PMID: 20103532
Authors: Azzu, V. - Brand, M. D.
Journal: J Cell Sci

Mitochondrial uncoupling protein 2 (UCP2) is implicated in a wide range of pathophysiological processes, including immunity and diabetes mellitus, but its rapid degradation remains uncharacterized. Using pharmacological proteasome inhibitors, immunoprecipitation, dominant negative ubiqbiquitiuitin mutants, cellular fractionation and siRNA techniques, we demonstrate the involvement of the ubiquitin-proteasome system in the rapid degradation of UCP2. Importantly, we resolve the issue of whether intramitochondrial proteins can be degraded by the cytosolic proteasome by reconstituting a cell-free system that shows rapid proteasome-inhibitor-sensitive UCP2 degradation in isolated, energised mitochondria presented with an ATP regenerating system, ubiquitin and 26S proteasome fractions. These observations provide the first demonstration that a mitochondrial inner membrane protein is degraded by the cytosolic ubiquitin-proteasome system.

post to: CiteULike

A putative role for microRNA-205 in mammary epithelial cell progenitors.

Publication Date: 2010 Feb 15 PMID: 20103531
Authors: Greene, S. B. - Gunaratne, P. H. - Hammond, S. M. - Rosen, J. M.
Journal: J Cell Sci

In an effort to understand the potential role of microRNAs (miRNAs) in mammary-gland stem or progenitor cells, miRNA microarrays were performed on subpopulations of the mouse mammary epithelial cell (MEC) line COMMA-DbetaGeo. This cell line contains a heterogeneous subpopulation of progenitors characterized by the expression of stem cell antigen 1 (Sca-1; encoded by Ly6a). Microarray analysis indicated that the Sca-1 subpopulations have distinct miRNA expression profiles. Functional studies were performed on miR-205, which was highly expressed in the Sca-1-positive (Sca-1(+)) cells. When miR-205 was overexpressed in vitro, the COMMA-DbetaGeo cells underwent several significant morphological and molecular changes. miR-205 overexpression led to an expansion of the progenitor-cell population, decreased cell size and increased cellular proliferation. In addition, the colony-forming potential of the two Sca-1 subpopulations was increased. Target prediction for miR-205 indicated that it might regulate the expression of the tumor-suppressor protein PTEN. Overexpression studies using reporter constructs confirmed that PTEN expression is regulated by miR-205. In addition to PTEN, several other putative and previously validated miR-205 targets were identified by microarray analysis, including the previously reported miR-205 targets ZEB1 and ZEB2. Additionally, in normal mouse MECs, high expression of miR-205 was observed in stem-cell-enriched cell populations isolated by FACS using established cell-surface markers.

post to: CiteULike

Membrane protein SMP-1 is required for normal flagellum function in Leishmania.

Publication Date: 2010 Feb 15 PMID: 20086045
Authors: Tull, D. - Naderer, T. - Spurck, T. - Mertens, H. D. - Heng, J. - McFadden, G. I. - Gooley, P. R. - McConville, M. J.
Journal: J Cell Sci

Eukaryotic flagella and cilia are surrounded by a membrane that is continuous with, but distinct from, the rest of the plasma membrane. In Leishmania parasites, the inner leaflet of the flagellar membrane is coated with the acylated membrane protein, SMP-1. Here, we provide evidence that SMP-1 stabilizes the flagellar membrane and is required for flagella elongation and function. The expression and flagella targeting of SMP-1 is tightly associated with flagella elongation during amastigote to promastigote differentiation. Deletion of the genes encoding SMP-1 and the flagellar pocket protein SMP-2, led to the production of short flagella and defects in motility. Alterations in the physical properties of the smp-1/smp-2(-/-) flagellar membrane were suggested by: (1) the accumulation of membrane vesicles in the flagellar matrix, and (2) further retraction of flagella following partial inhibition of sterol and sphingolipid biosynthesis. The flagella phenotype of the smp-1/smp-2(-/-) null mutant was reversed by re-expression of SMP-1, but not SMP-2. SMP-1 contains a jelly-roll beta-sheet structure that is probably conserved in all SMP proteins, and forms stable homo-oligomers in vivo. We propose that the SMP-1 coat generates and/or stabilizes sterol- and sphingolipid-rich domains in the flagellar membrane.

post to: CiteULike

Vinculin regulates cell-surface E-cadherin expression by binding to beta-catenin.

Publication Date: 2010 Feb 15 PMID: 20086044
Authors: Peng, X. - Cuff, L. E. - Lawton, C. D. - DeMali, K. A.
Journal: J Cell Sci

Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that vinculin is important for the organization of cell-cell junctions. However, this approach removes vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of vinculin, but not of a vinculin A50I substitution mutant that is defective for beta-catenin binding. These findings suggest that an interaction between beta-catenin and vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a beta-catenin mutant that fails to bind vinculin. Thus, our study identifies vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.

post to: CiteULike